ENLARGED VESTIBULAR AQUEDUCTS (EVA) Two mutated copies of the SLC26A4 gene are associated with Pendred syndrome, comprised of bilateral hearing loss with enlargement of the vestibular aqueduct (EVA) and thyroid goiter. Other EVA patients have a normal thyroid gland and only one mutated copy of SLC26A4. Our study previously identified the same combination, or haplotype, of noncoding sequence variants upstream of the non-mutated copy of SLC26A4 in most of these nonsydromic EVA patients. This haplotype defines the most common allele associated with hereditary hearing loss in Caucasians. In this reporting period, we showed that this haplotype, acting as a pathogenic Mendelian recessive allele is associated with a less severe phenotype than alleles with mutations affecting the coding regions or splice sites of SLC26A4. We have defined the cellular taxonomy and molecular transcriptomic architecture of the mouse endolymphatic sac using RNA-seq analysis of single cells isolated from the endolymphatic sac epithelium. We have identified two primary populations of cells in the mature mouse endolymphatic sac: mitochondria-rich cells (MRCs) and ribosome-rich cells (RRCs). Genes in which mutations cause EVA are differentially expressed in MRCs. The MRC transcriptome indicates that it is the primary cell type mediating sodium chloride absorption in the developing inner ear. This absorption of sodium chloride drives the absorption of water from the lumen of the sac. Disruption of this process leads to EVA and hearing loss. We ascertain families with multiple members with nonsyndromic EVA that is not associated with detectable SLC26A4 mutations or Pendred syndrome. Our hypothesis is that these families segregate recessive alleles at one or more other genetic loci that cause nonsyndromic EVA. We are using those families in a combinatorial linkage-based, functional candidate exome sequencing strategy to identify other genetic causes of EVA. We are prioritizing genes for analysis based upon differential expression in the MRCs of the developing endolymphatic sac . DFNA34 HEARING LOSS We mapped a novel nonsyndromic hearing loss locus, DFNA34, in a single large family. We used recombinations to define a critical map interval in which the gene and mutation must be located. We identified a likely mutation in a gene (NLRP3) in which other mutations cause hearing loss associated with autoinflammatory disease. In order to confirm this mutation as causative, we used massively parallel sequencing as well as conventional Sanger dideoxy sequencing to rule out mutations in any of the other genes in the critical map interval. We detect expression of the candidate gene, NLRP3, in the inner ear. We collaborated with Drs. Daniel Kastner. Paola Pinto-Patarroyo and Raphaela Goldbach-Mansky to study the patients for evidence of cochlear and systemic auto-inflammation on magnetic resonance imaging studies at the NIH Clinical Center. We hve detected evidence of systemic and cochlear auto-inflammation, providing conclusive proof of the pathogenic nature of the mutation we have detected. We showed the existence of macrophage/monocyte-like cells in the normal resting mouse cochlea. We showed that these cells are capable of expressing NLRP3 and secreting interleukin-1beta. Therefore the mouse cochlea has resident cells capable of mounting an innate immune response. We hypothesize that DFNA34 causes cochlear hearing loss by abnormal activation of the NLRP3 inflammasome pathway within the cochlea. We ascertained a second unrelated family with the exact same mutation segregating in the original DFNA34 family. The affected members of this second family also co-segregate signs and symptoms of auto-inflammation although they do not meet criteria for cryopyrin-associated periodic syndromes, a spectrum of auto-inflammatory disorders known to also be caused by NLRP3 mutations. Treatment with an IL-1beta blocker, anakinra, improved or resolved the hearing loss in 3 of 3 family members who were treated. The temporal bone imaging indicated that the cochlear auto-inflammation also improved or resolved, in correlation with their hearing test results. Therefore DFNA34 hearing loss is an example of a genetic form of hearing loss in which precise diagnosis can guide treatment which can stabilize or reverse the loss of hearing. We studied the natural history of hearing loss associated with an NRLP3 mutation and DFNA34. The hearing loss was delayed in onset and slowly progressive. TMC GENES We tested the hypothesis that expression of Tmc2 in the mature mouse cochlea could rescue the auditory phenotype associated with loss of Tmc1 in Tmc1-null mice. The results showed that Tmc2 could partially but not completely rescue hearing and inner ear sensory hair cell function and structure.